PROJECTSUMMARY Mitochondriaarecentersofmetabolismwhoseactivitiesneedtobecalibratedtomeetchangingcellularneeds. Generaldysfunctionoftheseorganellesisimplicatedinmanycommonhumandisorders,includingParkinson?s, Alzheimer?s, various cancers, metabolic syndrome, type 2 diabetes (T2D), obesity, non-alcoholic fatty liver disease(NAFLD),andheartfailure,mostoftenthroughunclearmeans.Definingthepathogenicmitochondrial alterationsthatcontributetothesemetabolicdisordersanddevisingnewtherapeuticstrategiestorectifythem representprincipalchallengesinmitochondrialmedicine.Apotentialcontributortothisdysfunctionisaberrant intra-mitochondrial protein phosphorylation?a process recognized as critical for pyruvate dehydrogenase regulationformorethan50years,butrelativelyunexploredotherwise.Recenteffortsfromourlaboratoriesand others have now revealed that mitochondrial proteins are replete with dynamic phosphorylation that changes reproduciblybetweenhealthyanddiseasedstates,andthatphosphorylationcanaltertheactivitiesofproteins involved in core metabolic pathways. We have also now connected select phosphorylation events to poorly characterized matrix protein phosphatases, thereby beginning to establish a mechanistic framework for understanding mitochondrial protein phosphorylation and its effects on metabolic activities. Given these emerging findings, the premise of this project is that reversible phosphorylation may be widely important in calibrating mitochondrial metabolism, and that its mismanagement could contribute to the pathophysiology of mitochondria-relateddisorders.Rigorousneweffortstorevealhowphosphorylationaffectsmitochondrialprotein functionandtodefinethephosphatasesthattargeteachsitemayultimatelyenableanewtherapeuticstrategy focusedonmanipulationofthemitochondrialphosphorylationnetwork.Theworkproposedhereisdesignedto take significant steps toward these goals. In particular, the contributions of our efforts will be 1) to define the physiologicalfunctionsanddirectbiochemicalsubstratesofPptc7,apoorlycharacterizedmitochondrialmatrix phosphatasewhosedisruptioncausesaseverefattyacidoxidation(FAO)-likedisorderandneonataldeath,2) toestablishthemechanisticeffectsofphosphorylationonputativePptc7substratesofoutstandingimportance to FAO and protein import, and 3) to begin systematically connecting the full set of orphan mitochondrial phosphatasestocandidatesubstratesandmetabolicprocesses,therebyopeningupalargelyuntappedareaof mitochondrialmetabolicregulation.Altogether,throughacomprehensiveapproachthatcombinesmammalian physiology, omics-level analyses, and rigorous biochemistry, we aim to make definitive connections between mitochondrialphosphatasesandtheirsubstrates,establishabroadframeworkforunderstandingtheroleofthis post-translation modification in calibrating mitochondrial activities, and ultimately pave the way for a new therapeuticstrategytorectifymitochondrialdysfunction.